After much effort, the aromatase enzyme, responsible for the conversion of androgens to estrogens, has been obtained in solubilized form, allowing the resolution of three distinct enzyme fractions, two of which have been reconstituted. We propose a series of detailed kinetic studies on these purified enzymes, emphasizing radiometric and isotope effect analysis. We have observed significant differences in enzyme functioning among the fractions, both in the relative quantity of intermediates produced and in the final products obtained. While probing the meaning of these differences, their existence will be exploited to optimize expression of the isotope effects associated with the three sequential oxidation steps. Our preliminary indications suggest that further investigation will pinpoint the site of controversial third oxidation, and elucidate the nature of the active enzyme-bound intermediate. We propose to assess the extent and origin of substrate specificity in the enzyme fractions and to detail the alterations in functioning observed in cross-reconstitution experiments when their reductase specificity is violated. The aromatization reaction is of central endocrinological importance due to the ubiquitous ramifications of estrogen production in areas ranging from reproductive physiology to cancer and brain gender-imprinting. The reaction is transferable in all essential details to the biosynthesis of other compounds with great medical and agrochemical importance including cholesterol, ergosterol, the major membrane sterol in fungi, and gibberellin, a key plant growth hormone. Elucidation of the reaction mechanism will give new insights into our understanding of steroid enzymology, cyt P-450 catalyzed drug oxidations and carcinogenesis, and toward designs of effective inhibitors for specific enzyme processes. The methods involve use of regio- and stereoselective isotope labeling of steroids, isotope distribution analysis, incubations with various enzyme preparations, isolation, identification and kinetic studies of intermediates and products, and isotope-effects analysis.